The CD45 antigen is a member of the protein tyrosine phosphatase (PTP) family and is a 180-240 kD trans-membrane glycoprotein. It is also known as the leukocyte common antigen (LCA), T200, or Ly-5. CD45 plays a key role in T-cell and B-cells receptor signal transduction. Different isoforms of CD45 exist due to variable splicing of its exons. These isoforms are very specific to the activation and maturation state of the cell as well as cell type. The various isoforms have the same trans-membrane and cytoplasmic segments, but different extra-cellular domains, and are differentially expressed on subpopulations of B- and T-cell lymphocytes. The primary ligands described for CD45 include galectin-1, CD1, CD2, CD3, CD4, TCR, CD22 and Thy-1.
Depending on which of the alternatively spliced exons (A, B or C) is recognized, antibodies restricted to recognizing one or the other isoform have been identified (termed CD45R). In addition, monoclonal antibodies (mAbs) binding an epitope common to all the different isoforms have also been identified. The mAbs designated CD45RA recognize the product of exon-A. The mAbs designated CD45RB recognize the product of exon-B. A third type of mAbs termed CD45RO (as exemplified by UCHL1) selectively bind to the 180 kD isoform (without any of the variable exons A, B or C) which is restricted to a subset of cortical thymocytes, activated T cells and memory cells, and is absent on B cells.
In general, all cells of hematopoietic origin, with the exception of mature erythrocytes and platelets, express CD45. High expression of CD45 is seen with most acute lymphoid and myeloid leukemias. Since CD45 is not found on tissues of non-hematopoietic origin, its specific expression in leukemia has made it a good target for developing therapeutics, including radio-immunotherapeutics. For example, CD45 is expressed at a density of approximately 200,000 to 300,000 sites per cell on circulating leukocytes and malignant B cells. One particular 131I-labeled anti-CD45 antibody, BC8, has been explored as a candidate radio-immunotherapeutic alone and in combination with chemotherapy or total body irradiation. The use of this 131I-anti-CD45 antibody for the treatment of subjects needing bone marrow transplant has also been explored.
A number of anti-CD45 antibodies are commercially available. These include human, mouse, rat, rabbit, canine antibodies and a host of related derived reagents, such as, conjugates with fluorophores, chromophores, biotin and dyes. These also include antibodies derived by the use of various epitopes, domains and regions of CD45 antigen. Several clones of the species-specific anti-CD45 antibody are also commercially available. A list of commercial suppliers of anti-CD45 antibodies include the following: eBioscience, Inc., San Diego, Calif., USA; Novus Biologicals, LLC, Littleton, Col., USA; Bethyl Laboratories, Inc., Montgomery, Tex., USA; AbD Serotec/Bio-Rad Inc., Raleigh, N.C., USA; BD Biosciences, San Jose, Calif., USA; AbCam Inc., Cambridge, Mass., USA; Enzo Life Sciences, Inc., Farmingdale, N.Y., USA; R&D Systems Inc., Minneapolis, Minn., USA; EXBIO Praha, A.S., Vestec, Czech Republic; Life Technologies, Grand Island, N.Y.; and many more. A comprehensive list of suppliers can be accessed at the following website:
http://www.biocompare.com/pfu/110447/soids/3537/Antibodies/CD45?vmpi_6408=1.
A search at this site for “CD45 antibody” performed on Sep. 25, 2014 came up with a total of 3,646 products that are available from 59 suppliers. It is evident that a number of anti-CD45 antibodies and related reagents are available with their own specific product characteristics such as species specificity and antigen heterogeneity, such as epitope or domain specificity.
While large numbers, varieties, and clones of anti-CD45 antibodies are available, none is structurally well described. For use as a therapeutic agent, it would be advantageous to properly describe an anti-CD45 antibody in order to develop the anti-CD45 antibody into a therapeutic for the treatment of human indications of hematopoietic malignancies.
Among several clones of the anti-CD45 murine antibody, BC8 recognizes all the human isoforms of the CD45 antigen. The potential applications of the BC8 antibody for clinical use, including the treatment of certain kinds of human lymphomas, are well defined in the literature of the past 20-25 years. Despite this, the structural composition and characterization of this isolated BC8 clone for use as a human therapeutic has not been adequately described.
Only a partial amino acid sequence from a BC8 clone has been described as being used in making a single chain construct with streptavidin. In this construct, certain regions corresponding to the putative variable regions of the light chain (LC) and heavy chain (HC) were fused together with the streptavidin sequence. Therefore, some 110-120 amino acid stretches related to the LC and HC of BC8 were used in this particular context which were indirectly identified by the methods of reverse transcription-PCR (RT-PCR).
As such, the complete structural composition of the BC8 mAb has not been described and characterized.
As indicated above, the BC8 antibody has been shown to bind to all the isoforms of human CD45, and thus provides an excellent target for the development of therapeutics for certain human malignancies of hematopoietic origin, including lymphomas. While radiolabelling of BC8 for targeted treatment of such malignancies has been explored, none of the prior art has been able to achieve radiolabelling efficiencies suitable for its efficient and cost effective use as a therapeutic. Furthermore, BC8 radiolabelled with 131I has a short half-life (the half-life of 131I is 8.02 days), and must be made and shipped to clinical sites on demand, which impedes its wider use as a therapeutic.
Radio-iodinated antibodies can undergo auto-radiolysis, due to radiation energy decay and associated free radical mechanisms. This can dramatically reduce the antibodies' immune-reactivity, rendering them less effective and thus unsafe for therapeutic use. Moreover, free radical reactivity towards certain amino acid residues (e.g., Tyr, Trp, His, Met and Cys) in a protein can affect the solution stability of a protein-based radiopharmaceutical composition. Thus, stabilizing target-specific radiopharmaceutical compositions remains a challenge in their development.
New compositions and methods are needed to stabilize 131I-anti-CD45 immunoglobulins, such as BC8, thereby prolonging shelf life and improving human therapeutic potential.